Intratumor microbiome: selective colonization in the tumor microenvironment and a vital regulator of tumor biology

Abstract The polymorphic microbiome has been proposed as a new hallmark of cancer. Intratumor microbiome has been revealed to play vital roles in regulating tumor initiation and progression, but the regulatory mechanisms have not been fully uncovered. In this review, we illustrated that similar to other components in the tumor microenvironment, the reside and composition of intratumor microbiome are regulated by tumor cells and the surrounding microenvironment. The intratumor hypoxic, immune suppressive, and highly permeable microenvironment may select certain microbiomes, and tumor cells may directly interact with microbiome via molecular binding or secretions. Conversely, the intratumor microbiomes plays vital roles in regulating tumor initiation and progression via regulating the mutational landscape, the function of genes in tumor cells and modulating the tumor microenvironment, including immunity, inflammation, angiogenesis, stem cell niche, etc. Moreover, intratumor microbiome is regulated by anti‐cancer therapies and actively influences therapy response, which could be a therapeutic target or engineered to be a therapy weapon in the clinic. This review highlights the intratumor microbiome as a vital component in the tumor microenvironment, uncovers potential mutual regulatory mechanisms between the tumor microenvironment and intratumor microbiome, and points out the ongoing research directions and drawbacks of the research area, which should broaden our view of microbiome and enlighten further investigation directions.


INTRODUCTION
Microorganisms are the important component of our body, with the cumulative microbial genome exceeding the human genome by a factor of more than 100. 1 With the help of next-generation sequencing (NGS), we are surprised to find that the microbiome also resides in the tumor tissue, even in those that do not directly connect with the external microenvironment. 2 Moreover, the microbiome would change along tumor initiation, 3 and bacteria in the primary tumor could be found in the metastatic tissues or even in the patient-derived xenograft (PDX) tissues after several successive passages. 4These results indicate the supportive force in the tumor microenvironment for the microbiome.The unique microenvironment within tumors may select certain species to reside.6][7][8] Of note, the role of microbiome in regulating the biology of cancer has recently been underscored, 9 and the polymorphic microbiome has been proposed as a new hallmark of cancer. 10Many researches have revealed the direct interaction between microbiome and tumor cells. 11As a newly found player in the tumor microenvironment, numerous studies have proven that the composition of tumor microbiota is significantly different from that of adjacent normal tissues 12 and is widely associated with clinical features of patients (Table 1).  Thesresults highlight the potential role of intratumor microbiome in regulating cancer development.Thus, the roles and mechanisms of mutual regulation among intratumor microbiome, tumor cells, and other components of the tumor environment urgently need to be uncovered.Moreover, the currently adopted anti-cancer therapies have been revealed to change the composition of intratumor microbiome, while intratumor microbiome could influence therapeutic effects.Thus, targeting or exploiting the intratumor microbiome has become the new research direction for better tumor control.
In this review, by comprehensively analyzing research results concerning intratumor microbiome, we presented the interplay between tumor cells and intratumor microbiome, mainly the bacteriome.We presented that as a component of the tumor microenvironment, the composition of intratumor microbiome is actively shaped by tumor cells and adopted anti-cancer therapies.The tumor microenvironment is hospitable for the colonization of certain microbiomes.And conversely, the intratumor microbiome contributes to genetic, epigenetic, and signaling changes in tumor cells, modulates the tumor microenvironment, and influences the efficacy of therapies.These facts highlight the vital role of intratumor microbiome in tumor initiation, progression, and therapy resistance, which could be a therapeutic target or exploited as a weapon to fight against cancer.Finally, we presented the ongoing research highlights and discussed the drawbacks in the research field of intratumor microbiome, in order to finally put forward the potential investigation directions in the future.In summary, this review emphasized that the interplay between intratumor microbiome and the tumor microenvironment, uncovered and summarized the underlying mechanisms, and presented the ongoing research directions and shortcomings in the research area, which could innovate more meaningful investigations.

THE COLONIZATION OF MICROBIOME IN THE TUMOR MICROENVIRONMENT
Innovated by the finding that Rous sarcoma virus caused sarcoma development in chicken, 34 oncologists have once spared no effort to search for onco-microorganisms.Although several microorganisms were revealed to be associated with cancer, 35 most cancers failed to be proven to be associated with microorganisms at that time, mainly due to the low biomass of microorganisms within cancer tissues.Many tissues from which the tumor arose were traditionally considered to be sterile.Thus, the existence of intratumor microbiome has been neglected and unrecognized for a long time.Luckily, with the advance of technologies, mainly NGS, it has been revealed that most tumor tissues contain microbiomes. 2Numerous studies have revealed differences in the composition of microbiome between healthy tissues and tumors (Table 1).Nevertheless, how and why the microbiome colonizes the tumor are not fully illustrated.In this review, we first systematically discussed the routes and mechanisms for the colonization of microbiome in tumor tissues (Figure 1).

Routes of the colonization of intratumor microbiome
Generally, there are several routes by which bacteria may colonize the tumor.First, tumors arise from the organs that directly connect with the external environment and may have bacteria from the resident microbiome; for example, nasopharyngeal microbiota was the main origin of intratumor bacteria in nasopharyngeal cancer. 14Especially, the tumor may disrupt the barrier of epithelium or mucosa, which could promote the colonization of resident microbiome.Tumors harboring TP53 mutations, which can impair epithelial function, have a unique bacterial consortium, mainly the Acidovorax temporans in lung cancer. 36 recent study revealed that the biopsy site, rather than primary tumor type, was associated with microbiome diversity, highlighting the role of adjacent environment.Second, bacteria may be transmitted from remote regions of our body to colonize tumor tissue via blood stream or other physical channels.Bacteria could be transmitted from the gut to pancreas as evidenced by Green Fluorescent Protein (GFP)-labeled Escherichia coli and Carboxyfluorescein Succinimidyl Ester (CFSE)-labeled Enterococcus faecalis, which were orally administered and detected in the pancreas. 38Same bacteria could be transmitted to tumor tissues via different mechanisms.For example, in colorectal cancer (CRC), both routes significantly contribute to the composition of intratumor microbiome.Barrier deterioration induced by CRC-initiating genetic lesions or inflammation results in the invasion of microbiota, which contributes to tumor development. 39ral-derived microbiome, including four Fusobacterium spp., was found to be enriched in colorectal tumors 40,41 ; although the reason why these bacteria translocated from the oral to gut was not fully illustrated, several studies have revealed the potential way.A previous study revealed that the common oral bacterium Fusobacterium nucleatum was more likely to translocate to CRC using the hematogenous route. 42Another study also revealed that oral gavage of F. nucleatum was sufficient to promote colorectal tumor development, suggesting that transmission via the gastrointestinal tract was also possible. 43

Hospitable tumor microenvironment for the colonization of intratumor microbiome
Despite the route by which the microbiome resides in tumor tissues, the selective colonization of certain bacteria may be ascribed to tumor tissues are permissive places for bacteria to reside and proliferate.When the bacteria Bifidobacterium breve was administered through tail vein injection or oral gavage, it could be specifically detected later in the subcutaneous tumor tissues. 44sing high-resolution in vivo bioluminescent imaging, after intravenous administration of bacteria including nonpathogenic commensal bacteria E. coli and B. breve, or Salmonella typhimurium, these bacteria were detected specifically in subcutaneous tumors of mice. 45These facts hint that the tumor tissue or certain microenvironmental factor could recruit specific species.Of note, the fact that F. nucleatum and its related bacteria were also detected in the metastatic CRC and PDX tissues after several successive passages indicated that the tumor microenvironment might be a "greenhouse" for certain bacteria. 4ore specifically, certain bacteria could colonize the tissue through the interaction of molecules between bacteria and tumor tissues.F. nucleatum colonized breast cancers via the Fap2-dependent binding to tumor-displayed Gal-GalNAc. 46Intravenously administrated F. nucleatum localized to mouse tumor tissues in a Fap2-dependent manner. 47However, most of the results displayed are found in F. nucleatum due to the intensive investigation of this bacteria.More results regarding the molecular interaction between intratumor microbiome and tumor cells will be found in the future as the research going forward.
Conversely, the bacteria could even help create a suitable microenvironment for themselves to reside in the tumor tissues.After systematic administration of Salmonella enterica serovar Typhimurium, tumor necrosis factoralpha was rapidly elevated in the blood, which significantly promoted bacteria to be flushed into the tumor followed by tumor necrosis formation, bacterial growth, and infiltration of neutrophilic granulocytes. 48

Factors that cause the similarities and differences in the intratumor microbiome
Overall, based on the biology of cancer and the characteristics of bacteria, the colonization of bacteria in the tumor may further ascribed to the following reasons.First, the tumor microenvironment is usually hypoxic, 49 which may potentiate the survival and proliferation of anaerobic and facultative anaerobic bacteria.In addition, different tissues may have different oxygen supply levels, which may account for the differences in the residing bacteria.Most cancers may select anaerobic bacteria but lung tumors could favor aerobic bacteria.Second, tumor tissues often have necrotic regions, and dying tumor cellderived components have been revealed to modulate the composition of microbiome, 50 the reason for which may be that the necrotic region could provide nutrients and other molecules for the bacteria to outgrowth.Third, tumor cells may release chemo-attractant compounds, which could recruit bacteria.Last, the tumor microenvironment is usually immune suppressive, which may protect the bacteria against the clearance by immune system. 513][54] In colon cancer models, depletion of neutrophils led to increased number of bacteria in tumors and proliferation of tumor cells accompanied by increased tumor cell DNA damage and an inflammatory response mediated by interleukin-17 (IL-17), 55 indicating that the intratumor microbiome is under the active surveillance of immune system and that the immune suppressive microenvironment may contribute to the colonization of bacteria.Conversely, neutrophilic inflammation seems to be permissive for tumor-promoting bacteria growth in squamous cell vulvar carcinoma, 33 indicating the contextdependent role of immune cells.As evidence, using in situ spatial-profiling technologies and single-cell RNA sequencing, it was revealed that bacterial communities populated microniches that were less vascularized and highly immuno-suppressive in oral squamous cell carcinoma and CRC. 56When comparing different tissue parts of oral cancers, the microbiome of tumor tissues had a greater alpha diversity, while minor differences in diversity existed between outer tumor tissues and inner tumor tissues.Nevertheless, Fusobacterium, Neisseria, Porphyromonas, and Alloprevotella were more abundant in outer tumor tissues, while Prevotella, Selenomonas, and Parvimonas were enriched in inner tumor tissues. 57

2.4
Risk factors for cancer influence the colonization of intratumor microbiome Furthermore, the microbiome could also be influenced by factors that are associated with the characteristics of patients, especially the known etiologies of cancers, indicating the important role of intratumor microbiome in potentiating tumor initiation.Intratumor microbiome in lung cancer was associated with age, gender, and other clinical features. 58And the abundance of A. temporans was also higher in smoking-associated lung cancers, indicating that smoking helps to create an environment for the growth of these bacteria and these bacteria may contribute to regulating the tumor biology. 36Patients with pancreatic cancer who were males and smokers showed the presence of potentially cancer-promoting or immune-inhibiting microbes in the pancreas, and had poorer prognosis. 59he etiologies of liver cancer, including alcohol and hepatitis B, significantly influenced the composition of the liver microbiome, which further participated in the tumorigenic process. 60Together, these data all support that the intratumor microbiome is influenced by many factors, and they could participate in the process of cancer progression.

MECHANISMS OF INTRATUMOR MICROBIOME IN MODULATING TUMOR INITIATION AND PROGRESSION
Numerous evidences have shown that the intratumor microbiome is associated with clinical features and prognosis of patients (Table 1).In the tumor microenvironment, the amount of microbiome was usually elevated, but the diversity of which was downregulated, with some bacteria species dominating the microenvironment.The effects of intratumor microbiome in promoting cancer development have been thoroughly discussed elsewhere, 61,62 and we herein will focus on the underlying mechanisms (Figure 2).

3.1
Intratumor microbiome modulates genetic changes and signaling in tumor cells

3.1.1
Intratumor microbiome induces genetic mutations and is associated with tumor genetics Genetic mutation is considered as the driving force of cancer development. 63,64Numerous researches have revealed that the microbiome directly induced DNA damage and thus accelerated tumor initiation.As a high-risk population for developing CRC, patients with familial adenomatous polyposis presented patchy bacterial biofilms composed predominantly of E. coli and Bacteroides fragilis, which encoded oncotoxins colibactin (clbB) and Bacteroides fragilis toxin (bft). 65Colibactin alkylates DNA with an unusual electrophilic cyclopropane. 66The bacteria in the bacterial biofilms induced IL-17 expression and DNA damage in colonic epithelium, which further led to faster tumor onset and increased mortality of patients. 65istinct mutational signatures were found before and after the exposure to pks + E. coli in colon organoids, and the signature after exposure was detected in patients from two independent cohorts, indicating that the underlying mutational process results directly from the previous exposure to pks + E. coli. 67However, findings also revealed that the majority of CRC cases did not have the mutational signature, and many patients who have pks + E. coli did not develop CRC.This is in consistence with the fact that the tumor development is a complex process influenced by many factors, and that the genetic mutation alone is not sufficient. 68Similarly, E. coli carrying the pks-islandproduced colibactin also induced a novel mutational signature in oral squamous cell carcinoma and in other mucosal tumor types. 69Furthermore, in oral and even upper aerodigestive tract carcinomas, the residing of certain bacteria, including Neisseria subflava, Streptococcus mitis, Candida albicans, Glabrata, etc., that are generally non-pathogenic, could convert alcohol into carcinogen acetaldehyde and thus play a subtle role in alcohol-related carcinogenesis in human oral cancer. 70,71Acetaldehyde causes DNA damage and blocks DNA repair, and is usually further metabolized by aldehyde dehydrogenase (ALDH) in the liver, while in the oral cavity, the activity of ALDH is relatively low, resulting in the long existence of acetaldehyde and more deteriorating results. 72,73Conversely, alcohol ingestion resulted in increased levels of Neisseria. 74And the fact that Neisseria is more abundant in outer tumor tissues 57 is also consistent with the role of Neisseria in promoting carcinogenesis.Moreover, pathogenic bacteria such as Bartonella henselae could transfer their conjugative DNA into nucleus of human cells by the VirB/VirD4 type IV secretion system, 75 which could be another source of genetic mutations.Taken together, these results reveal that the bacteria actively participate in modulating genetic changes and promoting cancer initiation via different mechanisms in different organs.
In addition, there are large amounts of evidences indicating that the composition of microbiome is associated with the genetic background of the tumor.By analyzing the mutational signature and intratumor microbiota in CRC, Okuda et al. found that Fusobacterium was associated with many mutated genes, as well as genes in cell cycle-related pathways. 76A high abundance of Campylobacter was associated with the mutational signature, including failure of double-strand DNA break repair. 76In biopsies with or without KRAS mutation or microsatellite instability (MSI), there was a significant difference in intratumor microbiota composition, 77 highlighting the association between intratumor microbial heterogeneity with genetic alteration.Moreover, significantly less microbial diversity was found in gastric cancer tissues compared with non-malignant tissues in both MSKCC and TCGA cohorts, and MSI-high gastric cancer had distinct microbial enrichment compared to other molecular subtypes. 78RC-associated intratumor genera, including Dialister and Casatella, were associated with the MSI status, 79 indicating the higher mutation rate associated with these bacteria.These facts further support the role of intra-tumor microbiome in regulating the tumor mutational landscape.

Intratumor microbiome modulates functions of genes
Besides directly accelerating genetic mutations, the intratumor microbiome could greatly influence the function of mutated genes, or even change their function, in tumor cells.The effects of mouse p53 hotspot mutations (R172H and R270H, corresponding to R175H and R273H in humans, respectively) on CRC models were controlled by the microbiota.In environments that lacked microbiota, p53 hotspot mutation exerted tumor suppressive effect, while in microenvironments presented with microbiota, the mutant p53 conversely exerted oncogenic properties. 80echanistical investigations revealed that this controversial effect was mediated by the metabolite derived from the microbiome-gallic acid, which reinstated the Transcription Factor-4 (TCF4)-chromatin interaction and the hyperactivation of Wnt signaling. 80Although similar researches concerning the role of microbiome in regulating gene functions are still lacking, this work strongly indicates that the role of intratumor microbiome should be noticed and worth further investigation.

Intratumor microbiome regulates cellular signaling
Intratumor microbiome could further modulate tumor cell signaling to potentiate cancer development.F. nucleatum was revealed to directly bind to E-cadherin, activate β-catenin signaling, and differentially regulate the inflammatory and oncogenic responses in CRC. 28And Peptostreptococcus anaerobius surface protein PCWBR2 directly interacted with colonic cancer cell lines via α 2 /β 1 integrin, which induced the activation of the PI3K-Akt pathway in CRC cells and led to increased cell proliferation and nuclear factor-kappa B (NF-κB) activation. 81ergara et al. further illustrated that microbiota-induced epithelial-mesenchymal transition (EMT) and inflammation were the common molecular mechanisms that induced tumor initiation and progression. 82In addition, in the muscle invasive bladder carcinoma, a variety of microbes, including E. coli, butyrate-producing bacterium SM4/1, and a species of Oscillatoria, were associated with the expression of classical EMT-associated genes, including E-cadherin, vimentin, SNAI2, SNAI3, and TWIST1. 83nd airway microbiota enriched in lung cancer patients, mainly the Streptococcus and Veillonella, contributed to the upregulation of the PI3K pathway in lung cancer. 84Interestingly, intratumor microbiome accompanied cancer cells during the metastasis process in breast cancer, and could promote cancer cell metastasis by enhancing cell viability through reorganization of the actin cytoskeleton. 85Intervention of tumor microbiota using antibiotics impeded tumor metastasis, but did not inhibit the growth of primary tumors. 85Besides, in oral cancer, the F. nucleatumsecreted outer membrane vesicles enhanced migration and metastasis of cancer cells via activating intracellular autophagy pathways. 86F. nucleatum infection in the pancreas induced the expression of cytokines, including Granulocyte-macrophage Colony-stimulating Factoe (GM-CSF), etc., from normal and cancerous pancreatic cells to promote the aggressive phenotype of pancreatic cancer cells. 87Together, these data reveal that the microbiome could regulate the signaling of cancer cells, which could be another mechanism that supports cancer development.

3.1.4
Intratumor microbiome influences the epigenetic status of cancer cells Moreover, there is also much evidence showing that microbiome regulates the biology of cancer cells via epigenetic mechanisms.In stomach cancer, the intratumor microbiome, especially Kytococcus sedentarius and Actinomyces orisinteracted, was strongly associated with methylation changes in immune-related genes of cancer cells and regulated gene expression. 88In esophageal cancer, the presence of F. nucleatum was associated with LINE-1 hypomethylation, which was associated with poor prognosis. 89Butyrate, a common metabolite of the gut microbiota, exerted antitumor effect via accumulating in the CRC cells due to the Warburg effect (due to the Warburg effect, butyrate is not consumed within the cancer cells) and thus functions as a histone deacetylase inhibitor to stimulate histone acetylation and affect apoptosis and cell proliferation. 90

Intratumor microbiome changes the metabolism of cancer cells
The intratumor microbiome can also modulate the tumor biology via influencing metabolism.In intrahepatic cholangiocarcinoma, the intratumor microbiota Paraburkholderia fungorum was significantly enriched in paracancerous tissues, which could inhibit tumor growth through alanine, aspartate, and glutamate metabolism. 91nd in breast cancer, integrating bulk and single-cell RNA sequencing data revealed that the intratumor microbiome was significantly associated with metabolic heterogeneity in breast cancers, highlighting the need for further mechanistic investigations. 92These results indicate that the intratumor microbiome may influence many aspects of cancer cells, but may differ between different cancer types; more specific and comparative investigations are thus needed.

Intratumor microbiome regulates the tumor microenvironment to potentiate tumor development
It has been widely recognized that tissue microenvironment plays vital roles in cancer initiation and progression. 68][95][96] Without suitable selective forces, mutated neoplasm cells remain dormant and could persist in the organ indefinitely without carcinogenesis. 97The microbiome in the tumor microenvironment also plays vital roles in inducing cancer carcinogenesis or metastatic cell outgrowth. 3,98Only when stroma is exposed to the carcinogen could they lead to the carcinogenesis of mammary tumors, regardless of whether the epithelial cells are exposed to the carcinogen or not. 99The tumor environment is one of the determining factors in inducing cancer initiation and progression, and numerous researches have revealed the role of intratumor microbiome in regulating the tumor microenvironment (Figure 2).

Intratumor microbiome enhances inflammation to promote tumor progression
Inflammation is one of the driving forces of carcinogenesis and metastatic colonization in various tissues, 100,101 and the microbiome significantly contributes to the establishment of an inflammatory microenvironment.In tet2-deficient mice, a model of spontaneous hematopoietic malignancies, the development of pre-leukemic myeloproliferation was highly dependent on microbial-induced inflammation. 102icrobiota-induced inflammation changed the epigenetic status of host cells, which further accelerated the process of inflammatory bowel disease and colitis-associated CRC. 103Moreover, tumor-resident bacteria E. coli further disrupted the gut vascular barrier (GVB) depending on the virulence regulator VirF.Then, bacteria disseminated to the liver, boosted the formation of a premetastatic niche, and favored the recruitment of metastatic cells. 104This phenomenon was validated in a clinical cohort.Patients with increased levels of PV-1, a marker of impaired GVB, were associated with liver bacteria dissemination and metachronous distant metastases. 104These facts reveal the important role of microbiome-related inflammation in cancer initiation and progression.

Intratumor microbiome regulates the tumor immune microenvironment
The cancer immune microenvironment is the most well-studied area of the interaction between intratumor microbiome and the tumor microenvironment.The ovarian cancer could be divided into immune-enriched and immune-deficient subtypes, which are correlated with the intratumor microbiome.And intratumor microbiome is closely associated with the prognosis of patients. 105e immune histopathological parameters such as PD-L1 expression and tumor-infiltrating lymphocytes were significantly associated with alpha and beta diversity of intratumor microbiome. 37Interestingly, the bacteria-specific peptides were presented by tumor cells in both Human Leukocyte Antigen (HLA)-I and HLA-II molecules.T cells that specifically recognize these bacterial antigens were identified in the tumor microenvironment.Recurrent bacterial peptides in tumors from different patients, as well as in different tumors from the same patient, were identified. 106These findings suggest that bacteria actively participate in modulating the tumor immunogenicity and tumor microenvironment and highlight a therapeutic direction.
Furthermore, the intratumor microbiome is highly associated with the immune landscape of the tumor tissues.Multi-omics analysis proved that intratumor microbiome was highly associated with tumor immune landscape and respones to immune therapy. 107In gastric cancer, elevated intratumor Methylobacterium was significantly associated with poor prognoses and reduced CD8 + tissue-resident memory T cells. 108In cutaneous melanoma, certain intratumor bacteria were associated with the infiltration of CD8 + cells and chemokines, including CXCL9, CXCL10, and CCL5, which were further correlated with overall survival (OS) of the patients. 32Microbial dysbiosis was also revealed to contribute to colon tumor susceptibility by hyperstimulating CD8 + T cells to promote chronic inflammation and early T-cell exhaustion, which further reduced antitumor immunity. 109As the rising star of immune checkpoint molecules, PD-L1 expression contributes to tumor immunosuppression and is a target for immune checkpoint blockade therapy.F. nucleatum in the CRC could enhance PD-L1 expression by activating the STING signaling, which enhanced the antitumor effects of PD-L1 blockade. 110And consistently, F. nucleatum in the CRC was also associated with T-cell infiltration and the MSI status, which was further differentially related to immune response. 111,112Nevertheless, most of these findings reveal the correlation but not the cause-effect mechanisms.Based on limited evidence that microbiome actively shapes the tumor microenvironment, we tend to believe that the intratumor microbiome is not just a bystander, and further research is highly needed.
Of note, the microbiome has been revealed to influence many types of immune and inflammatory cells in the microenvironment.The commensal microbiota promoted lung cancer development via stimulating Myd88dependent IL-1β and IL-23 production from myeloid cells, inducing the proliferation and activation of γδ T cells that produced IL-17 and other effector molecules to promote inflammation and tumor cell proliferation. 3The abundant bacteria in pancreatic cancer tissues were also revealed to generate a tolerogenic immune program by differentially activating Toll-like receptors in monocytic cells 38 ; ablation of these bacteria attenuated the oncogenesis and progression of pancreatic cancer and enabled the efficacy of checkpoint-targeted immunotherapy. 38Of note, F. nucleatum in the CRC tissues increased tumor multiplicity and selectively recruited tumor-infiltrating myeloid cells, which generated a proinflammatory microenvironment that can promote tumor progression. 43And periodontal pathogen Porphyromonas gingivalis could promote colorectal tumorigenesis by recruiting myeloid cells and creating a proinflammatory tumor microenvironment. 113imilarly, in oral squamous cell carcinoma and CRC, single-cell in situ spatial-profiling technologies and singlecell RNA sequencing showed that intratumor bacteria, including Fusobacterium and Treponema, enhanced cell heterogeneity and promoted cell migration and metastasis by changing cellular signaling and recruiting myeloid cells to bacterial regions. 56More specifically, F. nucleatum Fap2 could bind to human TIGIT and inhibit immune activity of natural killer (NK) and T cells. 114In addition, the colonization of F. nucleatum in CRC suppressed the accumulation of tumor infiltrating T cells and promoted tumor growth and metastatic progression, which could be suppressed by antibiotic treatment. 46And elevated P. gingivalis promoted pancreatic cancer initiation and progression by creating inflammatory microenvironment through recruiting neutrophils, which promoted cancer progression through releasing neutrophil elastase. 115Together, these results strongly support that intratumor microbiome actively regulates the tumor immune microenvironment to further contribute to cancer initiation and progression.

Intratumor microbiome modulates status of macrophages
As an important component of tumor microenvironment and a vital regulator of tumor biology, 116 macrophages are also regulated by the intratumor microbiome.In pituitary neuroendocrine tumors, intratumor bacteria were associated with more abundant microglia and the macrophages in nerve tissues, which further showed longitudinally branched morphology, 117 indicating the regulatory role of intratumor microbiome on macrophages.Similarly, more abundantly infiltrated M2 macrophages were detected in bacteria-dominant subtype in HBV-related hepatocellular carcinoma, which further showed multiple upregulated metabolism pathways. 118Specifically, absence of beneficial bacteria with ureolytic capacity, such as Bifidobacterium, was accompanied by elevated urea cycle metabolism in colorectal tumor tissue; the urea then enter into macrophages and further skew macrophages toward a pro-tumoral phenotype characterized by the accumulation of polyamines. 119Gut microbiome imbalance induced by dominant colonization of E. coli promoted the expression of CTSK, which then induced M2 macrophage polarization and CRC progression. 120Similarly, F. nucleatum in CRC promoted CCL20 expression in tumor cells, which further induced M2 macrophage polarization and enhanced the metastasis of cancer cells. 121Remotely, the indole-producing bacteria in the gut microbiome-derived indole compounds activated the AhR receptor in tumorassociated macrophages to suppress tumor growth and intra-tumoral infiltration of IFNγ + CD8 + T cells in pancreatic cancer. 122On the other hand, supplementation with Akkermansia muciniphila, a bacteria that was significantly reduced in CRC, suppressed colonic tumorigenesis and tumor progression by inducing the enrichment of M1-like macrophages in an NLRP3-dependent manner. 123hese facts reveal the regulatory role of intratumor microbiome in regulating the infiltration and differentiation of the macrophages, which further contribute to regulating tumor biology.

Intratumor microbiome influences angiogenesis
Besides, microbiome also participates in modulating the angiogenesis process. 124Dysregulated angiogenesis is a hallmark of cancer and contributes to tumor progression. 125Targeting the abnormal angiogenesis signaling has shown some therapeutic effects in cancer. 126uring the development of normal tissue, microbes colonizing the mucosal surface are assigned the responsibility for regulating elaboration of the underlying microvasculature by signaling through bacteria-sensing epithelial cells. 127As a vital onco-bacteria, Helicobacter pylori was revealed to be associated with increased Vascular Endothelial Growth Factor (VEGF) expression and neoangiogenesis, which may contribute to H. pylori-related gastric carcinogenesis. 128Quorum sensing peptides produced by diverse commensal or pathogenic bacteria could promote tumor cell invasion and angiogenesis, thereby potentially influencing tumor metastasis. 129In addition, exposure of cryptic-like intestinal epithelial cells to E. coli induced Hypoxia-inducible Factor 1 alpha (HIF-1α) protein expression, which further enhanced the expression of VEGF, a ligand that promotes angiogenesis. 130

3.2.5
Intratumor microbiome regulates stem cell niche Importantly, the intratumor microbiome also actively contributes to regulating cancer stem cell niche.During early life after birth, gut microbiota colonization, especially Lactobacillus, is vital in regulating stem cell niche and orchestrate stem cell differentiation and tissue development via influencing intestinal macrophages. 131In CRC, CRC-enriched microbiota metabolite isovalerate elevated 5-HT production in intestinal nerve cells, which constituted the stem cell niche to enhance the stemness of cancer stem cells and promoted CRC progression. 132Similar effect was also found in physiological stem cell self-renewal. 133. nucleatum-derived formate drove CRC tumor invasion by triggering AhR signaling and increasing cancer stemness. 134espite the great research advances of intratumor microbiome in regulating the tumor microenvironment, we should be aware that most of researches focused on the immune microenvironemnt, and few researches uncovered the specific mechanisms.The tumor microenvironment is rather complex and is composed of many components.The microbiome may also interact with the other components, such as fibroblasts, etc., which worths further investigations.

Cancer therapy modulates the composition of intratumor microbiome
While treating cancers, the adopted cancer therapeutic regimens could significantly change the microenvironment, including the intratumor microbiome (Figure 3A).After pancreaticoduodenectomy, the composition of microbiome in fecal samples, pancreatic fluid, bile, and jejunal contents was significantly changed, with enriched Klebsiella and Bacteroides and depleted anaerobic taxa (e.g., Roseburia and Faecalibacterium). 135These changes indicate that the intratumor microbiome could also be changed based on the former discussion that the resident microbiome is the important source of intratumor microbiome.Biliary stent placement and neoadjuvant chemotherapy with a combination of gemcitabine and paclitaxel were associated with significantly greater abundance of microbiota from the family Enterobacteriaceae in the tissue. 136urthermore, the bacteria were found to exploit dying cellreleased nutrients to accelerate growth.Dying cells, mainly apoptotic cells induced by several apoptotic triggers, accelerated the growth of pathogenic Salmonella via secreting pyruvate, which was mediated by the pyruvate formatelyase-encoding pflB gene. 508][139] It may be that the effect of dying tumor cells is partially mediated by the changed intratumor microbiome.Taken together, these results reveal that the cancer therapeutic strategies could change the composition of microbiome, which may partly be due to the change in the tumor microenvironment induced by the therapy.These changes may further influence the efficacy of therapy.However, related studies are relatively few, and further research is of high value.

Intratumor microbiome actively influences the efficacy of cancer therapy
Intratumor microbiome has recently been revealed to be associated with or to directly influence the therapy response by many studies.The metabolic ability and the metabolites of microbiome are vital in regulating therapeutic effects.Moreover, the regulation of microbiome on tumor cell biology also contributes to tumor therapy response (Figure 3B).
The effects of many chemotherapeutic drugs including gemcitabine, fludarabine, cladribine, etc. were revealed to be attenuated or enhanced by the bacteria that were commonly found in tumor tissues.These effects were at least partly mediated by the modification of chemical structure of drugs by bacteria. 140Further researches revealed that intratumor Gammaproteobacteria conferred gemcitabine resistance to cancers, including colon cancer, pancreatic cancer, etc., by expressing the bacterial enzyme cytidine deaminase (CDD L ).And most of the pancreatic cancer tissues have been detected with bacteria, the majority of which are Gammaproteobacteria, 141 indicating the underlying mechanisms of poor response to chemotherapy in pancreatic cancer.On the other hand, microbiota-derived tryptophan metabolite indole-3-acetic acid enhanced the chemotherapeutic effects of pancreatic cancer via elevating the accumulation of Reactive Oxygen Species (ROS) and downregulating autophagy in cancer cells. 142And intratumor Clostridium butyricum and its metabolite butyrate could promote susceptibility to ferroptosis in pancreatic cancer, 143 which is a promising way to enhance therapeutic effects. 144In breast cancer, probiotic Lactobacillus reuteri could translocate to intratumor tissues, which catalyzed the dietary tryptophan into I3A and promoted interferon-γ (IFN-γ)-producing CD8 T cells, thereby bolstering the effect of immune checkpoint inhibitors. 145These results highlight the important role of bacteria-derived metabolite in regulating therapy responses.
Moreover, F. nucleatum in CRC activated TLR4 to enhance autophagy in cancer cells and thus conferring chemoresistance. 146Intratumor lipopolysaccharide (LPS)- The interaction between intratumor microbiome and cancer therapy.(A) The adopted cancer therapy strategies may regulate the composition of intratumor microbiome, including directly eliminating bacteria by chemotherapy and changing the physicochemical microenvironment by surgery or other methods, and therapy-induced dying tumor cells may regulate the microbiome via secreting signal molecules or cellular components.(B) The intratumor microbiome may further influence therapy efficacy by directly degrading the chemotherapeutic agents or secreting metabolites.Moreover, the role of intratumor microbiome in regulating tumor biology may further contribute to influencing the efficacy of cancer therapy.activated NF-κB-IL6-STAT3 axis facilitated prostate cancer proliferation and docetaxel chemoresistance, which was caused by antibiotic use that elevated the abundance of Proteobacteria and gut permeability. 147Furthermore, the influence of microbiome in regulating the efficacy of immunotherapy was highly appreciated. 148Certain microbes showed the potential to predict the response to immune checkpoint blockade therapy with an AUC of 89%. 107Although most of the research concerning immunotherapy was conducted in gut microbiome, the potential effects of intratumor microbiome should be noted due to the aforementioned fact that the intratumor microbiome actively regulates the immune landscape.

EXPLOITING THE INTRATUMOR MICROBIOME AS THERAPEUTIC TARGETS
Since the intratumor microbiome plays vital roles in regulating tumor cell biology and the tumor microenvironment as well as the therapy response, selectively modulating the tumor microenvironment has become a research interest.Moreover, the tumor microenvironment is hospitable for the residing of certain bacteria; thus, engineering the bacteria to exploit them as therapeutic weapons has also shown promising potentials.However, due to the great diversity existing among different kinds of tumors, it is almost impossible to target the intratumor microbiome using an identical method.Thus, there are numerous attempts to manipulating the intratumor microbiome.

Targeting the intratumor microbiome by antibiotics
As most of the microbiome within tumors are pro-tumoral, the first attempt to control the influence of microbiome is applying antibiotics, which have shown some beneficial effects.Treatment with antibiotic metronidazole reduced Fusobacterium load in PDX tumor-bearing mice, and the proliferation of cancer cells and overall tumor growth were also inhibited after using antibiotics. 4Prolonged antibiotic treatment-induced microbiota diversity reduction was associated with higher intratumor immune response and a better antitumor effect induced by neoantigen cancer vaccines. 149And many experimental investigations have revealed the beneficial effects of antibiotic use. 3,46,85,98owever, in a prospective multicenter cohort study, prior antibiotic therapy impaired the efficacy of immune checkpoint inhibitor therapy (Hazard Ratio (HR), 7.4; 95% confidence interval [CI], 4.3-12.8;p < 0.001) and shortened the survival time of patients (HR, 7.4; 95% CI, 4.2-12.9). 150Prior to the immune checkpoint inhibitor era, antimicrobial prescription was associated with inferior overall and cancer-specific survival in breast cancer patients. 151And systematical use of antibiotics in gemcitabine chemotherapy of pancreatic cancer patients led to more severe side effects, including increased risk of anemia, thrombocytopenia, leukopenia, neutropenia, and gastrointestinal adverse events. 152These facts highlight the need for caution during intervention of bacteria using antibiotics.4][155] And the disruption of gut microbiome by antibiotics may otherwise lead to tumor progression or therapy resistance.Disruption of gut microbiota using antibiotics enhanced epithelial ovarian cancer progression and cisplatin resistance; metabolomics analyses revealed that this was partly due to the disruption of gut microbiota-derived metabolites. 156Disruption of the gut microbiota impaired the response of subcutaneous tumors to CpG-oligonucleotide immunotherapy and platinum chemotherapy via modulating myeloid-derived cell functions in the tumor microenvironment. 157Of note, the condensed tumor microenvironment may not be suitable for antibiotics to penetrate well, such as chemotherapeutic agents.Thus, there may be gaps between experimental and clinical conditions.Hence, attempting either to selectively target specific pathogens without perturbing the physiological microbiome or to re-establish commensal communities is a promising direction. 158

Strategies of specific modulating the intratumor microbiome
The capacity of bacteriophages to kill specific bacteria and modulate immunity makes them suitable candidates to manipulate the tumor microbiome and enhance the efficacy of tumor control. 159However, due to differences in anatomic and the tumor microenvironment, the administration and consequences of bacteriophages may be different, but the related studies are still lacking.Supplementation with probiotics is a feasible way to modulate the intratumor microbiome, especially in CRC.A consortium of 11 bacterial strains from healthy human donor feces was capable of robustly inducing IFN-γ-producing CD8 + T cells in the intestine. 160Oral application of a mix of four Clostridiales strains, or single application of Roseburia intestinalis or Anaerostipes caccae, prevented and even successfully treated CRC as stand-alone therapy via CD8 + T-cell-dependent manner in mice. 161In addition, using prebiotics to potentiate the diversity of microbiome could also be a way to modulate the microbiome.A bilberry anthocyanin combo containing chitosan and low molecular citrus pectin enriched the subdominant species, increased both the concentration and the proportion of butyrate in feces and enhanced intratumor CD8 + T-cell infiltration, which led to the best control of tumor growth. 162Prebiotic changed the gut microbiota and thus enhanced the antitumor immunity. 163In addition, administration of postbiotics may have superiority in terms of safety relative to their parental live cells. 164Interestingly, high-salt diet enhanced the intratumor colonization of Bifidobacterium by elevating gut permeability, which promoted NK-cell-mediated antitumor immunity. 165However, we should be aware that these experiments were mainly performed on CRC, due to easy manipulation through the oral route.Other methods should be considered in other cancer types.Although these strategies mainly target the gut microbiome, they may indirectly influence or provide some clues to modulate the intratumor microbiome.Of note, systematical adiministration of Bifidobacterium bacteria would accumulate in the tumor tissues and stimulate STING signaling, which further increased the cross-priming of dendritic cells after anti-CD47 treatment. 166And oral consumption of Lactobacillus decreased tumor growth of triple-negative breast cancer in a syngeneic breast cancer model. 167These results highlight the feasibility of modulating intratumor microbiome via oral route.

Exploiting intratumor microbiome as a weapon to fight against cancer
In addition, taking advantage of the characteristics of intratumor microbiome to suppress cancer is also under investigation.Tumor-resident microbiota showed great tumor-homing potential; thus, they could be potentially tumor-targeting weapons.Some bacteria isolated from the tumors showed preferential growth and proliferation within a targeted tumor milieu, and effectively caused immune cells to infiltrate the tumor and provoked strong anti-cancer responses in various syngeneic mouse models. 168Similar effect was also used to generate bacteria with intratumoral release of nanobodies targeting PD-L1 and CTLA-4, and administration of the bacteria showed enhanced therapeutic response. 169Bacillus toyonensis is normally presented in healthy people but absent in the feces of CRC patients.B. toyonensis-derived hemolysin BL has strong effects on killing tumor cells by causing cell membrane disruption, blebbing, and leakage of cytoplasmic contents, which has promising effects in controlling CRC. 170 Neospora caninum inhibited B16F10 melanoma by activating potent immune responses and directly destroying cancer cells. 171Moreover, direct intratumor injection of bacteria has presented some enlightening facts, and is also a feasible way.Intratumor injection of attenuated strain of Clostridium novyi showed promising effects in suppressing brain tumor. 172Further clinical study showed the therapeutic potential of intratumor injection in treatment-refractory advanced solid tumors. 173However, the high risk of adverse effects should be noted. 173Interestingly, as the recurrent bacterial peptides in tumors being identified, 106 tumor-specific CD8 + T cells with a second Tcell receptor (TCR) that recognizes a bacterial antigen were generated, and treatment of solid tumors with the dualspecific T cells and intratumor injection of bacteria showed therapeutic potentials. 174Moreover, clinical study showed that intratumor injection of engineered bacteria could regulate the tumor signaling and tumor microenvironment. 175nd there are opportunities that may promote the abscopal effects. 176

Ongoing findings of ways to regulate intratumor microbiome
Moreover, modulating the intratumor microbiome via other mechanisms has been investigated.Administration of adhesive hydrogel incorporating silver nanoparticles (which inhibited the growth of bacteria competing with Peptostreptococcus) alongside the intratumor delivery of the bacterium P. anaerobius synergized with PD-1 inhibition greatly enhanced the efficacy of PD-1 inhibition. 177Interestingly, screening of small-molecule inhibitors revealed that many antineoplastic agents, including 5-FU, were the potential inhibitors of F. nucleatum, clearing of which should enhance the efficacy of CRC treatment.However, the 5-Fluorouracil (5-FU) was metabolized by intratumor bacteria, including E. coli, to relieve the effect of 5-FU, 178 indicating that the intratumor microbiome may have synergistic effects.In addition, modulation of microbiome by traditional medicine, such as tranditional Chinese medicine, has been noted 179,180 ; although the investigation concerning intratumor microbiome is still lacking, it is an interesting research area that worths more efforts.

DISCUSSION
Recently, numerous studies have revealed that bacteria could reside in the tumor microenvironment, even tumors that are not directly in contact with the external microenvironment.These intratumor microbiome have been validated to be associated with the prognosis of patients, and participated in modulating tumor initiation, progression and therapy response.In this review, we highlighted that the intratumor microbiome, like the other components in the tumor microenvironment, is actively shaped by the tumor cells and surrounding microenvironment and conversely contributes to regulating the tumor biology.Specifically, the characteristics of the tumor microenvironment, including hypoxia, immune suppression, etc., are specifically suitable for the colonization of microbiome, and the interaction between tumor microenvironment and bacteria, such as molecular binding or secretions from the tumor cells enhances the colonization of microbiome.Conversely, the microbiome promotes cancer development via modulating genetic mutational landscape, the phenotype of genes, cellular signaling, cell metabolism, etc., and reprogramming the tumor inflammatory, immune, and angiogenic microenvironment as well as cancer stem cell niche (Table 2).Of note, therapeutic strategies could change the composition of intratumor microbiome, which reversely influences the efficacy of these therapies.Based on these findings, targeting the intratumor microbiome or engineering the microbiome to fight against cancer has become a feasible way.Specifically, targeting the harmful bacteria or supplement of probiotics showed therapeutic potential.However, challenges exist in the lack of powerful strategies to selectively eradicate pathogens, and accompanied side effects should be noted.Taking advantage of the tumor-homing features of the intratumor microbiome, engineering them to specifically kill cancer cells or release effective molecules provide potential ways to control tumors, but there are relatively few related investigations.More targeted therapeutic strategies based on deciphering the genetic and molecular basis of tumor cell-microbiome interaction should be the future investigational direction.These results strongly support that the intratumor microbiome is the vital component of the tumor microenvironment and promotes cancer development.
Of note, as the important component of our body, the diversity of bacterial genes is 100 times greater than that of our genes, which contain numerous genes that do not exist in human cells and may extend the ability of human body. 181,182The metabolic capacity that the human cells do not possess and their metabolites may play vital roles in pathophysiological processes.For example, N-acyl amide synthase genes were enriched in gastrointestinal bacteria and the lipids that they encode interacted with G-proteincoupled Receptors (GPCRs) to regulate gastrointestinal tract physiology. 183Under the circumstance of cancer therapy, the Gammaproteobacteria bacterial enzyme CDD L in pancreatic cancer inactivated the chemotherapy agent gemcitabine. 141Moreover, 1952 uncultured candidate bacterial species were identified by metagenome sequencing, which substantially expanded the known species repertoire of the collective human gut microbiota, and these bacteria encoded hundreds of newly identified biosynthetic gene clusters and possessed a distinctive functional capacity. 184These results strongly support the great potential of microbiome in regulating tumor biology, but their capacity and the underlying molecular mechanisms are not fully recognized.Further research concerning the genetic and molecular basis of the microbiome is of high value.
In addition, besides bacteria reside directly in the tumor microenvironment, microbiota reside in gut also regulate the tumor microenvironment by secreting signal molecules directly or indirectly.Gut microbiota-derived STING agonists induced IFN-I production by intratumor monocytes to regulate macrophage polarization and NK cell-dendritic cell crosstalk and thus enhancing antitumor immunity. 185While gut microbiome-derived butyrate was found to inhibit STING signaling activation following radiotherapy, which abrogated radiotherapy-induced tumor-specific cytotoxic T-cell immune responses without directly protecting tumor cells from radiation, 186 highlighting the context-dependent role of different composition of microbiome.Commensal microbe-derived butyrate was revealed to induce the differentiation of colonic regulatory T cells, possibly via enhancing histone H3 acetylation in the promoter and conserving non-coding sequence regions of the Foxp3 locus. 187Gut microbiome-mediated primary-to-secondary bile acid conversion controlled the liver-selective antitumor effect via regulating CXCL16 expression in liver sinusoidal endothelial cells. 188These results also strongly support the remote regulatory role of the microbiome, even though they do not directly interact with tumor tissues.This reminds us that we should treat the tumor as a systematical disease and broaden our view when managing cancers.
Although recent researches have mainly focused on the bacteriome, the role of other microorganisms should also be noted.The recent few years have also witnessed that besides bacteria, other microorganisms are also presented in the tumor microenvironment and showed great potential in modulating cancer progression.0][191] In both pancreatic cancer patients and mouse models, the fungi were found to be enriched by about 3000-fold compared with the normal pancreas, with Malassezia spp. as the most abundant species.Malassezia accelerated tumor oncogenesis and progression in a mannose-binding lectin-dependent manner. 192Moreover, intratumor fungi were one of the driving forces of IL-33 secretion in pancreatic cancer, which recruited Th2 cells and innate lymphoid cells 2 to promote cancer progression. 193Of note, gut commensal F I G U R E 4 Potential future investigational directions concerning intratumor microbiome.The microbiome contains large amount of unrecognized genetic information and the molecular basis for the interaction between microbiome and the tumor cells has just begun to be uncovered.Thus, mining the underlying mechanisms is of great value.Moreover, the tumor microenvironment is significantly different between different tumor types or even subtypes; this may account for the varied composition and function of intratumor microbiome.Thus, deciphering the context-dependent messages is of great value.Furthermore, exploiting the tumor microbiome also shows promising effects in controlling cancer; engineering the microorganisms is a potential feasible way.Last, developing reliable methods to specifically upregulate or deplete certain microorganisms, such as Fusobacterium nucleatum, is also a direction worth trying.
fungi and bacteria might have opposite influences on antitumor immunity following radiation, with bacteria promoting the response and fungi reducing it. 194In soft tissue sarcoma, a strong positive correlation between viral relative abundance and NK cell infiltration was observed, and more NK infiltration was associated with superior metastasis-free and OS, 195 highlighting the potential roles of intratumor virus in tumor biology.And virus may bring another level of messages in the tumor microenvironment. 196These results remind us that other microorganisms also possess great potential in modulating tumor biology, but we should be aware that reliable investigational methods and results to illustrate the cause-effect relationship between tumor cells and these microorganisms are still lacking.This is an ongoing area that worths more efforts.
Nevertheless, there are several problems that restrain the development of the research area.First, the contamination of external bacteria is quite a problem during the long process from specimen collection to sequencing. 197,198tringent sterile procedure and suitable control should be adopted to get the most reliable results. 2 New detection methods to test the accuracy are a way to validate the results.For example, by adopting comprehensive histological imaging, it was found that bacteria in the glioma were mostly localized near nuclear membranes or in the intercellular space. 199In addition, the consistency of formalin-fixed paraffin-embedded (FFPE) tissues and fresh frozen tissues in retrieving the composition of core bacteria within breast tumors highlights the meaning and reliability of retrospective studies, 200 which could simplify the intratumor studies and accelerate the research progress by resuscitating massive messages embedded in FFPE.Moreover, most of the results just uncover the correlation between microbiome and cancers, but the cause-effect evidences are still lacking.Thus, we could not know whether the changes in the microbiome in the tumor microenvironment are the passengers during cancer development or actively participate in the process in many researches.Furthermore, the intratumor microbiome is also influenced by many factors.Race is an important factor influencing the composition of intratumor microbiome, which is also significantly associated with the different tumor biology. 201,202And diet could be an influencing factor in changing the composition of intratumor microbiome. 203Mediterranean diet consumption led to increased mammary gland Lactobacillus abundance compared with western diet-fed monkeys 204 ; while Lactobacillus was revealed to be downregulated in breast cancer tissues and acted as a potential antitumor microbe. 205 high-salt diet was revealed to enhance the intratumor colonization of Bifidobacterium by elevating the gut permeability. 165These phenomena indicate that intratumor microbiome is regulated by many factors, and more stringent control conditions should be adopted when performing studies; and the microbiome is amendable for management, and strategies other than killing the bacteria could be considered (Figure 4).
In summary, we demonstrated that intratumor microbiome is a vital component of the tumor microenvironment and interacts with tumor cells and the surrounding microenvironment, which influences tumor develop-ment and therapy response and is a therapeutic target (Figure 5).More stringent experimental conditions, developing context-dependent strategies to uncover the genetic, and molecular basis for better understanding of the intratumor microbiome is the basis for further investigations.

A U T H O R C O N T R I B U T I O N S
M.J. conceived and drafted the manuscript, drew the figures, and discussed the concepts of the manuscript.Z.Y. and J.D. conceived and drafted the manuscript, and discussed the concepts of the manuscript.A.Y. and W.C. supervised the process, provided valuable discussion and revised the manuscript.T.W., Z.J., Y.Y., and K.N. provided valuable discussion and revised the manuscript.All authors have read and approved the final manuscript.

33 F I G U R E 1
Mechanisms of tumor microenvironment in shaping the composition of intratumor microbiome.The intratumor microbiome could enter the tumor tissue via several routes.The adjacent tissue-resident microbiome could colonize the tumor tissue via the disrupted epithelial barrier induced by tumor-associated genetic mutations or inflammation (A).Moreover, the microbiome may colonize the tissue from the remote region via blood flow or physical channels.And the microbiome may select to specifically reside in the tumor tissue via molecular interactions (B).More importantly, the characteristics of the tumor microenvironment are hospitable for certain microbiome (C), resulting in the colonization of microbiome in tumor tissues as well as the organ-and tumor type-specific composition of intratumor microbiome.

F I G U R E 2
Mechanisms of intratumor microbiome in regulating the tumor microbiology.The intratumor microbiome could regulate the tumor biology via various mechanisms, including directly regulating cancer cells (A) and modulating the tumor microenvironment (B).The prototype bacteria that were found to modulate tumor biology are listed on the left of the arrow, while the revealed mechanisms or molecules mediating the effects are listed on the right.

F I G U R E 5
Interplay between intratumor microbiome and the tumor microenvironment.(A) Routes that microbiome enters the tumor microenvironment.Tumors that directly connect with the external microenvironment could be colonized by microbiome from the resident bacteria.Microbiome could also transmit from the blood stream or other physical channels.(B) The tumor microenvironment is hospitable for the colonization of microbiome.The characteristics of the tumor microenvironment select suitable bacteria to reside and the interaction of microbiome with the tumor microenvironment promotes microbiome colonization.(C) Intratumor microbiome promotes cancer initiation and progression.The intratumor microbiome promotes cancer development via inducing genetic and signaling changes and modulating the tumor microenvironment through various mechanisms as presented.(D) Mutual regulation of the intratumor microbiome and the adopted therapy.The cancer therapy strategies change the composition of intratumor microbiome, while microbiome also actively influences the efficacy of cancer therapy. 37

type Microbiome features or bacteria species Clinical significance Reference
Examples of the clinical significance of intratumor microbiome.
TA B L E 1 TA B L E 1 (Continued)

TA B L E 2
Mechanisms of intratumor microbiome in influencing the tumor biology.